The purpose of this project is to investigate the regulatory mechanisms of phosphofructokinase (PFK). Although regulation of this enzyme activity by various metabolites in vitro is well characterized, many in vivo observations are still not understood. We discovered an "activation factor" for PFK which is Beta-D-fructose-2,6-P2, and which appears to be the most important effector of PFK. The synthesis and degradaton of fructose-2,6-P2 and the control of its turnover will be investigated. Using isolated rat hepatocytes the effect of Alpha- and Beta-adrenergic agonists and antagonists on the level of fructose-2,6-P2 will be studied. We found an enzyme termed fructose-6-P,2-kinase which catalyzes the synthesis of fructose-2,6-P2, and this enzyme will be purified and characterized. We will search for an enzyme which is responsible for the degradation of this factor and if successful, this enzyme will be isolated and characterized. We will attempt to elucidate the control mechanisms of these enzymes. We will also determine the distribution of fructose-2,6-P2 in various tissues of rats as well as in diabetic rats. In order to elucidate the mechanism of action of fructose-2,6-P2 on purified muscle and liver PFKs, ligand binding to phospho and dephospho PFKs, conformational changes and its effect on association-dissociation reactions will be studied. In order to determine the physiological significance of the phosphorylation of muscle PFK in vivo, attempts will be made to correlate muscle contraction with phosphorylation using perfused rat muscle. If successful, we will study the biochemical mechanism of the reaction.